This invention relates generally to alleles of the human mu opioid receptor gene, along with products derived from such alleles. Also included herein are methods of diagnosing various susceptibilities using such alleles and determining treatment for certain diseases based upon the presence of specific alleles of the human mu opioid receptor gene, and various diseases or disorders related to physiological functions regulated by the hypothalamus pituitary adrenal axis (HPA) or the hypothalamus pituitary gonadal axis (HPG).
Opioid drugs have various effects on perception of pain, consciousness, motor control, mood, autonomic function, and can also induce physical dependence. The endogenous opioid system plays an important role in modulating endocrine, cardiovascular, respiratory, gastrointestinal functions, and immune functions. Opioids, either exogenous or endogenous, exert their actions by binding to specific membrane-associated receptors.
Examples of exogenous opioids presently known include, opium, heroin, morphine, codeine, fentanyl, and methadone, to name only a few. Moreover, a family of over 20 endogenous opioid peptides has been identified, wherein the members possess common structural features, including a positive charge juxtaposed with an aromatic ring that is required for interaction with an opioid receptor. It has been determined that most, if not all the endogenous opioid peptides are derived from the proteolytic processing of three precursor proteins, i.e., prbopiomelanocortin, proenkephalin, and prodynorphin. In addition, a fourth class of endogenous opioids, the endorphins, has been identified (the gene encoding these proteins has not yet been cloned). In the processing of the endogenous opioid precursor proteins, initial cleavages are made by membrane-bound proteases that cut next to pairs of positively charged amino acid residues, and then trimming reactions produce the final endogenous opioids secreted from cells in vivo. Different cell types contain different processing enzymes so that, for example proopiomelanocortin can be processed into different endogenous peptides by different cells. For example, in the anterior lobe of the pituitary gland, only corticotropin (ACTH), xcex2-lipotropin, and xcex2-endorphin are produced. Both pro-enkephalin and pro-dynorphin are similarly processed by specific enzymes in specific cells to yield multiple opioid peptides.
Pharnacological studies have suggested there are numerous classes of opioid receptors which bind to exogenous and endogenous opioids. These classes differ in their affinity for various opioid ligands and in their cellular and organ distribution. Moreover, although the different classes are believed to serve different physiological functions, there is substantial overlap of function, as well as of distribution.
In particular, there are at least three known types of opioid receptors, mu (xcexc), delta (xcex4), and kappa (xcexa), to which morphine, the enkephalins, and the dynorphins can bind. These three opioid receptor types are the sites of action of opioid ligands producing analgesic effects. However, the type of pain inhibited and the secondary functions vary with each receptor type. The mu receptor is generally regarded as primarily associated with pain relief, and drug or other chemical dependence, i.e., addiction and alcoholism.
The human mu opioid receptor, which modulates corticotropin releasing hormone, has been isolated and described in PCT Application WO 95/07983 (Mar. 23, 1995) (SEQ ID NO:1) as well as in Chen, Y., Mestek, A., Hurley, J. A., and Yu, L. (1993) Mol. Pharrnacol. 44, 8-12, and Wang, et al., FEBS letters, (1994)338:217-222. Furthermore, SEQ ID NO:1 an readily be obtained in GENBANK under accession number L25119. The cDNA herefor contains an open reading frame capable of encoding a protein of 400 amino acid residues with 94% sequence similarity to the rat mu opioid receptor. Hydropathy analysis of the deduced protein indicates the presence of seven hydrophobic domains, typical of G-protein-coupled receptors. The N-terminus contains five potential N-linked glycosylation sites which remain conserved between the human and the rat mu opioid receptor. A variant in which Asn-40 is changed to Asp (N40D) is reported in GENBANK Accession No. U12569.
In the body and brain, heroin is hydrolyzed to morphine, which acts at the mu opioid receptor and results in an euphoric effect and confers the reinforcing properties of the drug and contributes to development of addiction. Heroin addiction can be managed through treatment, primarily methadone maintenance. However, the biological basis of heroin addiction may include diversity of gene structure. Such genetic diversity of the human mu opioid receptor, and the impact of such diversity on receptor function, could contribute to the success or failure of pharmacological management. Similar problems with respect to patient response to pharmacological treatment could occur in most, if not all addictive diseases, such as heroin addiction, alcohol addiction, or cocaine addiction to name only a few, or a combination thereof.
Moreover, addiction to opioid drugs, especially heroin, is a major social problem in the United States, and throughout the world. For example, recent epidemiological assessments sponsored by the NIH-NIDA and other federal agencies have found that around 2.7 million persons in the United States have used heroin at some time. Moreover, the numbers of xe2x80x9chardcorexe2x80x9d long-term heroin addicts (addiction being defined herein as self administration of a regular, multiple, daily dose use of a short-acting opioid, such as heroin, for one year or more, with the development of tolerance, physical dependence and drug-seeking behavior, a definition codified in the Federal guidelines governing pharmacotherapy using long-acting agents such as methadone or LAAM, and used as the minimal requirement for entry into treatment) are now estimated to be approximately one million persons. In addition, it has been estimated that around 24 million persons in the United States have used cocaine for some time, and of that number, approximately one million use cocaine regularly, and at least 600,000-700,000 are cocaine addicts.
In view of the importance of the human mu opioid receptor in the study of addiction, and the epidemic proportions of drug addiction, especially to heroin, alcohol or cocaine, or a combination thereof, in the United States and throughout the world, and its involvement in the neuroendocrine system, and physiological functions regulated thereby, efforts have been made to investigate whether any polymorphisms in the gene encoding the human mu opioid receptor exist in the population, and whether such polymorphisms result in a phenotype that has an increased or decreased susceptibility towards development of addiction to exogenous opioids, such as heroin, or alcohol, cocaine, or other addictive drugs. For example, in an article entitled Human mu opioid receptor gene polymorphisms and vulnerability to substance abuse (Berrettini, W. H., Hoehe, M. R., Ferraro, T. N., DeMaria, P. A., and Gottheil, E., Addiction Biolog 2:303-308 (1997)), two polymorphisms in the human mu opioid receptor gene were reported. One polymorphism (G to T) occurs at nucleotide 175 preceding initiation of translation, and a second coding polymorphism(copyright) to T) at nucleotide 229 (with respect to transcription initiation) on exon I results in an Ala to Val residue change. However, data taken from a study indicated the C229T polymorphism does not differ in occurrence with statistical significance in addicts relative to non addicts (Id at 306). No functional studies were reported.
It has been further determined that a receptor for both endogenous and exogenous opioids modulates the activity of the hypothalamus pituitary adrenal axis (HPA) and the hypothalamus pituitary gonadal axis (HPG), which effects the neuroendocrine system and its production of signaling compounds that play important roles in regulation of numerous physiological functions. In particular, the neuroendocrine system involves the integration of the neural and endocrine systems of the body, and is responsible for the coordination of numerous bodily functions. An important part of this system is the hypothalamus, a specialized portion of the brain involved in receiving and relaying messages from the central nervous system to other parts of the body. Upon stimulation by chemical signals from the central nervous system, the hypothalamus secretes hypothalamic hormones, such as corticotropin releasing factor (CRF) or hormone and gonadotropin releasing hormone or luteinizing hormone releasing hormone. These factors in turn stimulate the anterior pituitary gland to secrete tropic hormones, or tropins, which are synthesized as relatively long polypeptides, and then are then biotransformed to produce active peptide hormones. Pro-opiomelanocortin, which is processed into several active peptide hormones, including adrenocorticotropic hormone (ACTH), is an example of a tropic hormone. ACTH stimulates the adrenal cortex to secrete additional hormones, like cortisol, a stress hormone in humans which regulates glucose metabolism, and targets many tissues in the body. In addition, examples of hormones produced by the anterior pituitary glad upon stimulation with gonadotropin releasing hormone include follicle-stimulating hormone and luteinizing hormones. These hormones stimulate the gonads, such as the ovaries and the testes, to secrete androgens, such as testosterone, progesterone, and estrogen, which in turn affect sexual development, sexual behavior, and other reproductive and nonreproductive functions. As a result, the endogenous opioid system plays an important role in modulating endocrine, reproductive, cardiovascular, respiratory, gastrointestinal, immune functions, sexual development and function, as well as a person""s response to stress.
More specifically, in humans, it has been determined that chronic administration of opioids has an inhibitory effect on the HPA axis [McDonald et al., Effect of morphine and nalorphine on plasma hydrocortisone levels in man. J. Pharmacol. Exp. Ther. 125:241247 (1959)]. Basal levels of ACTH and cortisol are significantly disrupted in active heroin addicts: suppression of ACTH and cortisol and abnormal diurnal rhythms of these hormones are found [Kreek, Medical safety and side effects of methadone in tolerant individuals. JAMA 223:665-668 (1973)]. Basal levels and the diurnal rhythm of ACTH and cortisol, which are disrupted in active heroin addicts, have been shown to become normalized in moderate to high dose, long-term methadone-maintained patients when compared to those of healthy volunteer subjects [Kreek, 1973; Kreek et al., Circadian rhythms and levels of beta-endorphin, ACTH, and cortisol during chronic methadone maintenance treatment in humans. Life Sci. 33:409-411 (1983); Kreek et al., Prolonged (24 hour) infusion of the opioid antagonist naloxone does not significantly alter plasma levels of cortisol and ACTH in humans. Proceedings of the 7th International Congress on Endocrinology Elsevier Science p1170, 1984].
In healthy volunteers, ACTH and cortisol levels decrease below the basal levels in response to the infusion of xcex2-endorphin indicating feedback of inhibition of pituitary ACTH release or suppression of hypothalamic CRF release by xcex2-endorphin [Taylor, et al., Beta-endorphin suppresses adrenocroticotropin and conisol levels in normal human subjects. J. Clin. Endocrinol. Metab. 57:592-596 (1983)], and also naloxone (an opioid antagonist) stimulates a rise in serum ACTH and cortisol, suggesting that the HPA axis is under the tonic inhibitory control of endogenous opioids normalized in steady-state chronic methadone-maintained patients; their HPA axis responses to metyrapone-induced stress appear to be no different from that of healthy volunteer subjects [Kreek, 1973; Kreek et al., Prolonged (24 hour) infusion of the opioid antagonist naloxone does not significantly alter plasma levels of cortisol and ACTH in humans. Proceedings of the 7th International Congress on Endocrinology Elsevier Science p1170, 1984].
Support for the effects of opioids on physiological functions regulated by the HPA and the HPG axes can be found in observations of heroin addicts. More specifically, it has been observed that many heroin addicts are infertile, and in the case of female addicts, their menstrual cycle is dramatically disrupted to the point that they do not ovulate. Furthermore, it has been observed that heroin addicts, and nonaddicted patients taking morphine, become constipated, and that the immune systems of addicts is weakened relative to the immune system of non addicts. However, once therapeutic agents designed to treat addiction, such as methadone, addicts become fertile, are no longer constipated, and have a immune system whose ability to fight foreign bodies is in parity with the immune system of a nonaddict.
Hence, what is needed is discovery of heretofore unknown polymorphisms of the human mu opioid receptor gene that can be used as genetic markers to map the locus of the human mu opioid receptor gene in the genome.
What is also needed are the DNA sequences of heretofore unknown isolated nucleic acid molecules which encode human mu opioid receptors, wherein the DNA sequences include a combination of presently known and subsequently discovered polymorphisms of the human mu opioid receptors.
Furthermore, what is needed is the characterization of the binding properties of heretofore unknown human mu opioid receptors produced from the expression of genes comprising such heretofore unknown polymorphisms of the human mu opioid receptor gene, or combinations of unknown polymorphisms and known polymorphisms.
Furthermore, what is needed is a characterization of the activity of such unknown human mu opioid receptors produced from the expression of nucleic acid molecules comprising such polymorphisms.
What is also needed is a correlation between polymorphisms of the human mu opioid receptor gene, and the susceptibility of a subject to addictive diseases, such as heroin addiction, cocaine addiction, or alcohol addiction, to name only a few.
What is also needed are diagnostic methods to determine a subject""s increased or decreased susceptibility to addictive diseases. With the results of such methods, targeted prevention methods, early therapeutic intervention, and improved chronic treatment to opioid addiction can be developed. Physicians armed with the results of such diagnostic methods can determine whether administration to a subject of opioid analgesics is appropriate or whether non-opioid derived analgesics should be administered to the subject. Also, appropriate choice and type of analgesic can be made in treating a subject""s pain.
What is also need are methods of determining a subject""s susceptibility to pain and responsibility to analgesics, and using that information when prescribing analgesics to the subject. What is also needed is an ability to determine the binding affinity of the mu opioid receptor to endogenous opioids, such as xcex2-endorphin, and the effect of this binding activity on the neuroendocrine system.
The citation of any reference herein should not be construed as an admission that such reference is available as xe2x80x9cPrior Artxe2x80x9d to the instant application.
There is provided, in accordance with the present invention, heretofore unknown polymorphisms of the human mu opioid receptor gene, and their use in mapping the locus of the human mu opioid receptor gene, determining susceptibility to addictive diseases, determining susceptibility to pain, and determining a therapeutically effective amount of pain reliever to administer to a subject suffering from pain, diagnosing a disease or disorder in a subject that is related to a physiological function regulated by the HPA or HPG axes of the neuroendocrine system, and selecting an appropriate therapeutic agent and a therapeutically effective amount of such an agent to administer to a subject suffering from a disease or disorder related to a physiological function regulated by the HPA or HPG.
Hence, the present invention extends to heretofore unknown polymorphisms of the human mu opioid receptor gene that can serve as genetic markers to map the locus of the human mu opioid receptor gene.
The present invention further extends to DNA sequences of heretofore unknown isolated nucleic acid molecules which encode human mu opioid receptors, wherein the DNA sequences include a combination of presently known polymorphisms and polymorphisms of the human mu opioid receptors discovered by Applicants.
The present invention further extends to the characterization of the binding properties of heretofore unknown human mu opioid receptors produced from the expression of isolated nucleic acid molecules comprising DNA sequences with such heretofore unknown polymorphisms of the human mu opioid receptor gene, or combinations of unknown polymorphisms and known polymorphisms.
Furthermore, the present invention extends to characterizing the activity of such unknown human mu opioid receptors and particularly the increased or decreased ability of mu opioid receptors produced from isolated nucleic acid acids of the present invention to activate G protein-activated inwardly rectifying K+ (GIRK) channels via a G protein-mediated mechanism.
The present invention further extends to Applicants"" discovery that polymorphisms in an allele comprising a DNA sequence of SEQ ID NO:1, such as A118G and C17T, which are described in further detail infra, are present in the population at a high frequency (greater than 5%).
Furthermore, the present invention extends to Applicant""s discovery of a correlation between polymorphisms of the human mu opioid receptor gene, and the increased or decreased susceptibility of a subject to addictive diseases, such as heroin addiction, cocaine addiction, or alcohol addiction, to name only a few.
The present invention further extends to diagnostic methods to determine a subject""s increased or decreased susceptibility to addictive diseases. With the results of such methods, targeted prevention methods, early therapeutic intervention, and improved chronic treatment to opioid addiction are set forth herein and encompassed by the present invention. In addition, attending medical professionals armed with the results of such diagnostic methods can determine whether administration of opioid analgesics is appropriate or whether non-opioid derived analgesics should be administered to the subject. Furthermore, appropriate choice and type of analgesic to treat a subject""s pain can be made.
Also, the present invention extends to methods of determining a subject""s increased or decreased susceptibility to pain and response to analgesics, and the use of the information in prescribing analgesics to the subject.
In addition, the present invention extends to methods of diagnosing a disease or disorder in a subject, wherein the disease or disorder is related to a physiological function regulated by the HPA or HPG axes of the neuroendocrine system. Examples of such physiological functions include reproductive or sexual functions, gastrointestinal motility, immune response, and ability to withstand stress.
Broadly the present invention extends to an isolated variant allele of a human mu opioid receptor gene which can serve as a genetic marker, wherein the predominant or xe2x80x9cmost commonxe2x80x9d allele of a human mu opioid receptor gene found in the population comprises a DNA sequence of SEQ ID NO:1, and a variant allele of the present invention comprises a DNA sequence having a variation in SEQ ID NO:1, wherein the variation comprises:
G24A;
G779A; or
G942A,
or combinations thereof.
Furthermore, the present invention extends to an isolated variant allele of a human mu opioid receptor gene as set forth above, which is detectably labeled. Numerous detectable labels have applications in the present invention, such as radioactive elements, chemicals which fluoresces, or enzymes, to name only a few.
The present invention further extends to an isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated variant allele of the human mu opioid receptor gene, wherein the predominant or xe2x80x9cmost commonxe2x80x9d allele of a human mu opioid receptor gene found in the population comprises a DNA sequence of SEQ ID NO:1, and a variant allele of the present invention comprises a DNA sequence having a variation in SEQ ID NO:1, wherein the variation comprises:
G24A;
G779A; or
G942A,
or combinations thereof.
Moreover, the present invention extends to an isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated variant allele of the human mu opioid receptor gene, wherein the predominant or xe2x80x9cmost commonxe2x80x9d allele of a human mu opioid receptor gene found in the population comprises a DNA sequence of SEQ ID NO:1, and a variant allele of the present invention comprises a DNA sequence having a variation in SEQ ID NO:1, wherein the variation comprises:
G24A;
G779A; or
G942A,
or combinations thereof, wherein the isolated nucleic acid molecule is detectably labeled. Examples of detectable labels that have applications in this embodiment of the present invention are described above.
In addition, the present invention extends to an isolated variant allele of a human mu opioid receptor gene, wherein the predominant or xe2x80x9cmost commonxe2x80x9d allele of the human mu opioid receptor gene encodes a human mu opioid receptor comprising an amino acid sequence of SEQ ID NO:2, and the variant allele of the human mu opioid receptor gene encodes a variant human mu opioid receptor comprising an amino acid sequence having a variation in SEQ ID NO:2, wherein the variation comprises Arg260His.
Furthermore, the present invention extends to an isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated variant allele of a human mu opioid receptor gene of the present invention, wherein the isolated nucleic acid molecule encodes a variant human mu opioid receptor comprising an amino acid sequence having a variation in SEQ ID NO:2, wherein the variation comprises Arg260His.
Naturally, the present invention extends to a variant human mu opioid receptor comprising an amino acid sequence having a variation in SEQ ID NO:2, wherein the variation comprises Arg260His.
Furthermore, the present invention extends to an antibody having a variant human mu opioid receptor comprising an amino acid sequence having a variation in SEQ ID NO:2, wherein the variation comprises Arg260His as an immunogen. Such an antibody can be a polyclonal antibody, a monoclonal antibody, or a chimeric antibody. Moreover, an antibody of the present invention can be detectably labeled. Examples of detectable labels which have applications in this embodiment comprises a radioactive element, a chemical which fluoresces, or an enzyme, to name only a few.
In addition, the present invention extends to cloning vectors that can be used to clone copies of a variant alleles of a human mu opioid receptor gene of the present invention. For example, the present invention extends to a cloning vector comprising an isolated variant allele of a human mu opioid receptor gene and an origin of replication, wherein the predominant or xe2x80x9cmost commonxe2x80x9d allele of a human mu opioid receptor gene found in the population comprises a DNA sequence of SEQ ID NO:1, and a variant allele of the present invention comprises a DNA sequence having a variation in SEQ ID NO:1, wherein the variation comprises:
G24A;
G779A; or
G942A,
or combinations thereof.
In another embodiment, the present invention extends to a cloning vector comprising an isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated variant allele of a human mu opioid receptor gene, and an origin of replication, wherein the predominant or xe2x80x9cmost commonxe2x80x9d allele of a human mu opioid receptor gene found in the population comprises a DNA sequence of SEQ ID NO:1, and a variant allele of the present invention comprises a DNA sequence having a variation in SEQ ID NO:1, wherein the variation comprises:
G24A;
G779A; or
G942A,
or combinations thereof.
Numerous cloning vectors have applications in the present invention. For example, a cloning vector having applications in the present invention includes E. coli, bacteriophages such as lambda derivatives, plasmids such as pBR322 derivatives, and pUC plasmid derivatives such as pGEX vectors or pmal-c or pFLAG, to name only a few.
Naturally, the present invention extends to expression vectors comprising an isolated variant allele a human mu opioid receptor gene operatively associated with a promoter, wherein the predominant or xe2x80x9cmost commonxe2x80x9d allele of a human mu opioid receptor gene found in the population comprises a DNA sequence of SEQ ID NO:1, and a variant allele of the present invention comprises a DNA sequence having a variation in SEQ ID NO:1, wherein the variation comprises:
G24A;
G779A; or
G942A,
or combinations thereof.
Furthermore, the present invention extends to an expression vector comprising an isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated variant allele a human mu opioid receptor gene, wherein the isolated nucleic acid molecule is operatively associated with a promoter. As set forth above, the predominant or xe2x80x9cmost commonxe2x80x9d allele of a human mu opioid receptor gene found in the population comprises a DNA sequence of SEQ ID NO:1, and a variant allele of the present invention comprises a DNA sequence having a variation in SEQ ID NO:1, wherein the variation comprises:
G24A;
G779A; or
G942A,
or combinations thereof
Numerous promoters have applications in an expression vector of the present invention, including but not limited to immediate early promoters of hCMV, early promoters of SV40, early promoters of adenoviris, early promoters of vaccinia, early promoters of polyoma, late promoters of SV40, late promoters of adenovirus, late promoters of vaccinia, late promoters of polyoma, the lac the trp system, the TAC system, the ARC system, the major operator and promoter regions of phage lambda, control regions of fd coat protein, 3-phosphoglycerate kinase promoter, acid phosphatase promoter, or promoters of yeast a mating factor, to name only a few.
In addition, the present invention extends to a unicellular host transformed or transfected with an expression vector of the present invention. Examples of hosts which can be transformed or transfected with an expression vector of the present invention, and have applications in the present invention, include, but are not limited to, E. coli, Pseudomonas, Bacillus, Streptomyces, yeast, CHO, R1.1, B-W, L-M, COS1, COS7, BSC1, BSC40, BMT10 or Sf9 cells.
Naturally, the present invention extends to a method of producing a variant human mu opioid receptor comprising an amino acid sequence having a variation in SEQ ID NO:2, wherein the variation comprises Arg260His. An example of such a method comprises the steps of culturing a unicellular host transformed or transfected with an expression vector comprising an isolated variant allele a human mu opioid receptor gene, wherein the predominant or xe2x80x9cmost commonxe2x80x9d allele of a human mu opioid receptor gene found in the population comprises a DNA sequence of SEQ ID NO:1, and a variant allele of the present invention comprises a DNA sequence having a variation in SEQ ID NO:1, wherein the variation comprises G779A, operatively associated with a promoter. The transformed or transfected unicellular host is then cultured under conditions that provide for expression of the variant allele of the human mu opioid receptor gene. The variant human mu opioid receptor produced from such induced expression is then recovered from the unicellular host.
Another example comprises the steps of culturing a unicellular host transformed or transfected with an expression vector comprising an isolated nucleic acid molecule operatively associated with a promoter, wherein the isolated nucleic acid molecule is hybridizable under standard hybridization conditions to a variant allele a human mu opioid receptor gene, and the predominant or xe2x80x9cmost commonxe2x80x9d allele of a human mu opioid receptor gene found in the population comprises a DNA sequence of SEQ ID NO:1, and the variant allele comprises a DNA sequence having at least one variation in SEQ ID NO:1, wherein the at least one variation comprises G779A. The transformed or transfected unicellular host is then cultured under conditions that provide for expression of the variant allele of the human mu opioid receptor gene. The variant human opioid receptor produced from such induced expression is then recovered from the unicellular host.
Furthermore, the present invention extends to an isolated variant allele of a human mu opioid receptor gene, wherein the predominant or xe2x80x9cmost commonxe2x80x9d allele of the human mu opioid receptor gene comprises a DNA sequence of SEQ ID NO:1, and a variant allele of the present invention comprises a DNA sequence having at least two variations in SEQ ID NO:1, wherein the variations comprise:
A118G;
C17T;
G24A;
G779A; or
G942A.
The present invention further extends to an isolated variant allele of a human mu opioid receptor gone comprising a DNA sequence having at least two variations in SEQ ID NO:1, as stated above, which is detectably labeled. Examples of detectable labels having applications in this embodiment include, but are not limited to, a radioactive element, a chemical which fluoresces, or an enzyme.
The present invention further extends to an isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated variant allele of a human mu opioid receptor gene, wherein the predominant or xe2x80x9cmost commonxe2x80x9d allele of the human mu opioid receptor gene comprises a DNA sequence of SEQ ID NO:1, and a variant allele of the present invention comprises a DNA sequence having at least two variations in SEQ ID NO:1, wherein the variations comprise:
A118G;
C17T;
G24A;
G779A; or
G942A.
Naturally, the present invention extends to a detectably labeled isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated variant allele of a human mu opioid receptor comprising a DNA sequence having at least two variations in SEQ ID NO:1, wherein the variations comprise:
A118G;
C17T;
G24A;
G779A; or
G942A.
Examples of detectable labels having applications in this embodiment of the invention include, but are not limited to, a radioactive element, a chemical which fluoresces, or an enzyme.
Furthermore, the present invention extends to an isolated variant allele of a human mu opioid receptor gene comprising a DNA sequence having at least two variations in SEQ ID NO:1, as set forth above, wherein the predominant or xe2x80x9cmost commonxe2x80x9d allele of a human mu opioid receptor gene encodes a human mu opioid receptor comprising an amino acid sequence of SEQ ID NO:2, and a variant allele of the present invention encodes a human mu opioid receptor comprising an amino acid having at least two variations in SEQ ID NO:2, wherein the variations comprise:
Asn40Asp or conserved variants thereof;
Ala6Val or conserved variants thereof; or
Arg260His or conserved variants thereof
The present invention further extends to an isolated nucleic acid molecule hybridizable under standard hybridization conditions to an isolated variant allele of a human mu opioid receptor gene comprising a DNA sequence having at least two variations in SEQ ID NO:1, wherein the variations comprise:
A118G;
C17T;
G24A;
G779A; or
G942A,
such that the isolated nucleic acid molecule encodes a variant human mu opioid receptor comprising an amino acid sequence having at least two variations in SEQ ID NO:2, wherein the variations comprise:
Asn40Asp or conserved variants thereof;
Ala6Val or conserved variants thereof; or
Arg260His or conserved variants thereof.
Naturally, the present invention extends to a variant human mu opioid receptor comprising an amino acid sequence having at least two variations in SEQ ID NO:2, wherein the variations comprise:
Asn40Asp or conserved variants thereof;
Ala6Val or conserved variants thereof; or
Arg260His or conserved variants thereof.
Moreover, the present invention extends to an antibody having as an immunogen a human mu opioid receptor comprising an amino acid sequence having at least two variations in SEQ ID NO:2, wherein the variations comprise:
Asn40Asp or conserved variants thereof,
Ala6Val or conserved variants thereof; or
Arg260His or conserved variants thereof.
An antibody of the present invention can be a polyclonal antibody, a monoclonal antibody, or a chimeric antibody. Moreover, an antibody of the present invention can be detectably labeled. Examples of detectable labels having applications in an antibody of the present invention include, but are not limited to, a radioactive element, a chemical which fluoresces, or an enzyme.
Furthermore, the present invention extends to a cloning vector comprising an isolated variant allele of a human mu opioid receptor gene and an origin of replication, wherein the predominant or xe2x80x9cmost commonxe2x80x9d allele of the human mu opioid receptor gene present in the population comprises a DNA sequence of SEQ ID NO:1, and a variant allele of the present invention comprises a DNA sequence having at least two variations in SEQ ID NO:1, wherein the variations comprise:
A118G;
C17T;
G24A;
G779A; or
G942A.
In addition, the present invention extends to a cloning vector comprising an isolated nucleic acid molecule hybridizable under standard hybridization conditions to a variant allele of a human mu opioid receptor and an origin of replication, wherein the variant allele comprises a DNA sequence having at least two variations in SEQ ID NO:1, wherein the variations comprise:
A118G;
C17T;
G24A;
G779A; or
G942A,
and an origin of replication.
Numerous cloning vectors have applications in this embodiment of the present invention. Examples of such vectors include, but are not limited to, E. coli, bacteriophages, such as lambda derivatives, plasmids such as pBR322 derivatives, and pUC plasmid derivatives such as pGEX vectors or pmal-c or pFLAG, to name only a few.
Naturally, the present invention extends to an expression vector comprising an isolated variant allele of a human mu opioid receptor gene operatively associated with a promoter, wherein such an isolated variant allele comprises a DNA sequence having at least two variations in SEQ ID NO:1, wherein the variations comprise:
A118G;
C17T;
G24A;
G779A; or
G942A.
In addition, the present invention extends to an expression vector comprising an isolated nucleic acid molecule operatively associated with a promoter, wherein the isolated nucleic acid molecule is hybridizable under standard hybridization conditions to an isolated variant allele of a human mu opioid receptor gene comprising a DNA sequence having at least two variations in SEQ ID NO:1, wherein the variations comprise:
A118G;
C17T;
G24A;
G779A; or
G942A.
Numerous promoters are available and have applications in an expression vector of the present invention. Examples of promoters having applications include, but are not limited to immediate early promoters of hCMV, early promoters of SV40, early promoters of adenovirus, early promoters of vaccinia, early promoters of polyoma, late promoters of SV40, late promoters of adenovirus, late promoters of vaccinia, late promoters of polyoma, the lac the trp system, the TAC system, the TRC system, the major operator and promoter regions of phage lambda, control regions of fd coat protein, 3-phosphoglycerate kinase promoter, acid phosphatase promoter, or promoters of yeast xcex1 mating factor, to name only a few.
Naturally, the present invention extends to a unicellular host transformed or transfected with an expression vector of the present invention. Examples of unicellular hosts having applications in an embodiment of the present invention include, but are not limited to, E. coli, Pseudonomas, Bacillus, Streptomyces, yeast, WHO, R1.1, B-W, L-M, COS1, COS7, BSC1, BSC40, BMT10 or Sf9 cells.
In another embodiment, the present invention extends to a method for producing a human mu opioid receptor comprising an amino acid sequence having at least two variations in SEQ ID NO:2, wherein the variations comprise
Asn40Asp or conserved variants thereof;
Ala6Val or conserved variants thereof; or
Arg260His or conserved variants thereof.
More specifically, an example of a method for producing such a human mu opioid receptor comprises the steps of culturing a unicellular host transformed or transfected with an expression vector comprising an isolated variant allele of a human mu opioid receptor gene operatively associated with a promoter, wherein the variant allele comprises a DNA sequence having at least two variations in SEQ ID NO:1, wherein the variations comprise:
A118G;
C17T;
G24A;
G779A; or
G942A,
under conditions that provide for expression of the isolated variant allele of a human mu opioid receptor gene. After expression, a variant human mu opioid receptor is recovered from the unicellular host.
In another example, a method for producing a human mu opioid receptor of the present invention comprises the steps of culturing a unicellular host transformed or transfected with an expression vector comprising an isolated nucleic acid molecule operatively associated with a promoter, wherein the isolated nucleic acid molecule is hybridizable under standard hybridization conditions to an isolated variant allele of a human mu opioid receptor gene comprising a DNA sequence having at least two variations in SEQ ID NO:1, wherein the variations comprise:
A118G;
C17T;
G24A;
G779A; or
G942A,
under conditions that provide for expression of the isolated nucleic acid molecule. The variant human mu opioid receptor produced from the expression is then recovered from the unicellular host.
Moreover, Applicants have discovered that xcex2-endorphin, an endogenous opioid comprising 31 amino acid residues, and binds to the human mu opioid receptor, has a binding affinity approximately three times greater for a variant human mu opioid receptor produced from expression of a variant allele of the human mu opioid receptor gene comprising an A118G variation in SEQ ID NO:1, than for a human mu opioid receptor produced from expression of the predominant or xe2x80x9cmost commonxe2x80x9d allele of the human mu opioid receptor gene. Also, xcex2-endorphin induced activity of a receptor produced from the expression of a variant allele of a human mu opioid receptor gene or an isolated nucleic acid molecule hybridizable thereto under standard hybridization conditions comprising the A118G polymorphism effected the receptor""s activation of GIRK channels via a and protein-mediated mechanism relative to the activity of a receptor produced from the expression of the predominant or xe2x80x9cmost commonxe2x80x9d allele. xcex2-endorphin is present in both the central nervous system (CNS) and the periphery. It plays a role in endogenous analgesia, as well as in response to exposure to a potential addictive agent, such as heroin or alcohol. For example, as a neuropeptide, it can modulate neurotransmitter actions in the CNS to mediate antinoception. It is also of potential importance for the pathophysiology of addictive diseases. Given the diverse roles of xcex2-endorphin, the presence of a variant allele of a human mu opioid receptor gene comprising at least one variation in SEQ ID NO:1, wherein the variation comprises A118G, in a subject may alter the subject""s perception of pain, susceptibility to develop opioid addiction following exposure to opioids as well as addictions to other drugs that alter the opioid system, and reaction of the subject towards a therapeutic agent designed to treat pain, such as morphine, or towards a therapeutic agent designed to treat a specific addiction. In fact, the inventors herein have also discovered that the A118G variant polymorphism is present in the human mu opioid receptor gene of at least one subset of nonaddicts, an Hispanic study subject group, in a statistically significant level (P=0.0041). Hence, the presence of the A118G polymorphism in a subject, with increased xcex2-endorphin affinity and effects on activation of GIRK channels as described above, is expected to decrease the subject""s perception of pain, protect the subject against potential addiction to opioids such as heroin and likewise influence the subject""s reaction to therapeutic agents designed to treat the at least one addictive disease of the subject.
Furthermore, Applicants have discovered a variant allele of a human mu opioid receptor gene comprising a variation in SEQ ID NO:1, wherein the variation comprises C17T, is present at a statistically significant greater frequency in the genome of at least one defined subset of addicts suffering from at least one addictive disease, than in the genomes of people not suffering from such a disease. Hence, the presence of such a variant allele of a human mu opioid receptor gene may alter perception of pain, susceptibility to develop opioid addiction following exposure to opioids, and influence the subject""s reaction to therapeutic agents designed to treat the at least one addictive disease of the subject. The inventors herein have discovered that the C17T variant mu opioid receptor has a endorphin binding affinity approximately 0.72 times that of the human mu opioid receptor produced from expression of the predominant or xe2x80x9cmost commonxe2x80x9d allele of the human mu opioid receptor gene. The decreased binding affinity of this variant is in contrast to the increased affinity of the A118G variant mentioned above. In addition, one of the important indices of cellular functioning of the mu opioid receptor is the inhibition of adenylyl cyclase activity following agonist binding to the receptor. The inventors herein have discovered that the C17T variant mu opioid receptor is less sensitive to two endogenous opioids, xcex2-endorphin and leu-enkephalin, than the receptor produced by the xe2x80x9cmost commonxe2x80x9d allele: the EC50 values differed by a factor of approximately 2.7 and 2.8, respectively. Hence, the presence of the C17T polymorphism in a subject, with decreased Fendorphin affinity and decreased sensitivity to endogenous opioids as described above, is expected to increase the subject""s perception of pain, increase susceptibility to potential addiction to opioids such as heroin and likewise influence the subject""s reaction to therapeutic agents designed to treat the at least one addictive disease of the subject.
Accordingly, the present invention extends to a method for determining a susceptibility in a subject to at least one addictive disease, comprising the steps of removing a bodily sample comprising a first and second allele of a human mu opioid receptor gene from the subject, and determining whether the first allele comprises a human mu opioid receptor gene comprising a DNA sequence having at least one variation in SEQ ID NO:1, wherein the variation comprises:
A118G; or
C17T.
The present of at least one of these variations in the human mu opioid receptor gene of the first allele is expected to be indicative of the subject""s susceptibility to at least one addictive disease relative to the susceptibility of a standard to at least one addictive disease, wherein the standard comprises a first allele comprising a human mu opioid receptor gene having a DNA sequence of SEQ ID NO:1.
Another embodiment of the method for determining a susceptibility in the subject to at least one addictive disease, as described above, comprises the further step of determining whether the second allele of the bodily sample of the subject comprises a human mu opioid receptor gene comprising a DNA sequence having at least one variation in SEQ ID NO:1, wherein the variations comprise:
A118G; or
C17T.
The presence of at least one variation the second allele of the bodily sample is expected to be indicative of the subject""s susceptibility to at least one addictive disease relative to a standard in which both alleles of a human mu opioid receptor gene comprise a DNA sequence of SEQ ID NO:1.
In particular, the presence of an A118G variation in the DNA sequence of the human mu opioid receptor gene of the first and/or second alleles in the bodily sample from the subject is expected to be indicative of a decreased susceptibility of the subject to at least one addictive disease relative to the standard.
Moreover, the presence of a C17T variation in the DNA sequence of the human mu opioid receptor gene of the first and/or second alleles in the bodily sample from the subject is expected to be indicative of an increased susceptibility of the subject to at least one addictive disease relative the susceptibility of the standard to at least one addictive disease, wherein both alleles of the standard comprise a human mu opioid receptor gene comprising a DNA sequence of SEQ ID NO:1. Examples of at least one addictive disease includes, but is not limited to opioid addiction, cocaine addiction or addiction to other psychostimulants, nicotine addiction, barbiturate or sedative hypnotic addiction, anxiolytic addiction, or alcohol addiction.
In another embodiment, the present invention extends to a method for determining a susceptibility to at least one addictive disease in a subject relative to susceptibility to at least one addictive disease in a standard, involving the detection of variations in the human mu opioid receptor itself, and particularly, determining whether a variant human mu opioid receptor is present in a bodily sample from a subject. Such a method comprises the steps of removing a bodily sample comprising a human mu opioid receptor from the subject, and determining whether the human mu opioid receptor present in the sample is a variant human mu opioid receptor of the invention, wherein the variant human mu opioid receptor comprises an amino acid sequence having at least one variation in SEQ ID NO:2, wherein the variation comprises:
Asn40Asp or conserved variants thereof; or
Ala6Val or conserved variants thereof,
The presence of at least one variation is expected to be indicative of the subject""s susceptibility to at least one addictive disease relative to susceptibility to at least one addictive disease in a standard, wherein the human mu opioid receptor of the standard comprises an amino acid sequence of SEQ ID NO:2.
In particular, a variant human mu opioid receptor present in the sample comprising an amino acid sequence having at least one variation in SEQ ID NO:2, wherein the variation comprises Asn40Asp or conserved variants thereof, is expected to be indicative of a decreased susceptibility to at least one addictive disease in the subject relative susceptibility to the at least one addictive disease in the standard, wherein the human mu opioid receptor of the standard comprises an amino acid sequence of SEQ ID NO:2.
In contrast, a variant human mu opioid receptor present in a sample from the subject comprising a variation in SEQ ID NO:2, wherein the variation comprises Ala6Val or conserved variants thereof, indicates an increased susceptibility to addictive diseases in the subject relative to a standard having a human mu opioid receptor comprising an amino acid sequence of SEQ ID NO:2.
As explained above, at least one addictive disease includes, but is not limited to, opioid addiction, cocaine addiction or addiction to other psychostimulants, nicotine addiction, barbiturate or sedative hypnotic addiction, anxiolytic addiction, or alcohol addiction.
Furthermore, the present invention extends to a method for determining a susceptibility to pain in a subject relative to susceptibility to pain in a standard, comprising the steps of removing a bodily sample comprising a first and second allele of a human mu opioid receptor gene from the subject, and determining whether the first allele comprises a human mu opioid receptor gene comprising a DNA sequence having at least one variation in SEQ ID NO:1, wherein the variation comprises:
A118G; or
C17T.
The presence of at least one variation in the human mu opioid receptor gene of the first allele is expected to be indicative of a decreased or increased susceptibility to pain in the subject relative to susceptibility to pain in the standard, wherein the first allele of the standard comprises a human mu opioid receptor gene comprising a DNA sequence of SEQ ID NO:1.
Moreover, a method for determining a susceptibility to pain in a subject may further comprise the step of determining whether the second allele comprises a human mu opioid receptor gene comprising a DNA sequence having at least one variation in SEQ ID NO:1, wherein the variation comprises:
A118G; or
C17T.
The presence of the at least one variation in the human mu opioid receptor gene of the second allele of the bodily sample from the subject is expected to be indicative of an increased or decreased susceptibility to pain in the subject relative to the susceptibility to pain in the standard, wherein the second allele in the standard comprises a human mu opioid receptor gene comprising a DNA sequence of SEQ ID NO:1.
At least one variation in the human mu opioid receptor gene of the first and/or second allele of the bodily sample taken from the subject, wherein the variation comprises A118G, is expected to be indicative of a decreased susceptibility to pain in the subject relative susceptibility of pain in the standard, wherein the human mu opioid receptor gene of the first and/or second allele of the standard comprises a DNA sequence of SEQ ID NO:1.
Furthermore, the presence of at least one variation comprising C17T in the human mu opioid receptor gene of the first and/or second allele of the bodily sample from the subject is expected to be indicative of an increased susceptibility to pain in the subject relative to the susceptibility to pain in the standard, wherein the first and/or second allele of the standard comprise a human mu opioid receptor gene comprising a DNA sequence of SEQ ID NO:1.
In another embodiment, the present invention extends to a method for determining a susceptibility to pain in a subject relative to susceptibility to pain in a standard by examining a bodily sample taken from the subject for the presence of a variant human mu opioid receptor. Such a method comprises the steps of removing a bodily sample comprising a human mu opioid receptor from the subject, and determining whether the human mu opioid receptor present in the sample is a variant human mu opioid receptor of the invention, i.e., comprises an amino acid sequence having at least one variation in SEQ ID NO:2, wherein the variation comprises:
BAsn40Asp or conserved variants thereof; or
Ala6Val or conserved variants thereof,
such that the presence of at least one variation is expected to be indicative of the subject""s susceptibility to pain relative to susceptibility to pain in the standard, wherein the human mu opioid receptor of the standard comprises an amino acid sequence of SEQ ID NO:2.
In particular, the presence of a variant human mu opioid receptor comprising an amino acid sequence having at least one variation in SEQ ID NO:2 wherein the variation comprises Asn40Asp or conserved variants thereof, is expected to be indicative of a decreased susceptibility to pain in the subject relative to susceptibility to pain in the standard, wherein the human mu opioid receptor of the standard comprises an amino acid sequence of SEQ ID NO:2.
Furthermore, the presence of a variant human mu opioid receptor comprising an amino acid sequence having a variation in SEQ ID NO:2, wherein the variation comprises Ala6Val or conserved variants thereof, in a bodily sample taken from a subject is expected to be indicative of an increased susceptibility to pain in the subject relative to susceptibility to pain in the standard, wherein the human mu opioid receptor of the standard comprises an amino acid sequence of SEQ ID NO:2.
Once a susceptibility to pain in the subject has been determined, it is possible for attending medical professionals treating the subject to administer to an appropriate, or therapeutically effective amount of pain reliever in order to induce analgesia in the subject. Administration of such an amount is important to the subject because, should an inappropriate amount of pain reliever be administered, the subject may not experience analgesia, and may be exposed to potentially deleterious side effects of the pain reliever, such as induction of addiction to the pain reliever, brain damage, or death.
Consequently, the present invention extends to a method for determining a therapeutically effective amount of pain reliever to administer to a subject in order to induce analgesia in the subject relative to a therapeutically effective amount of the pain reliever to administer to a standard in order to induce analgesia in the standard, wherein the method comprises determining a susceptibility to pain in the subject relative to susceptibility to pain in the standard. The susceptibility of pain in the subject is expected to be indicative of the therapeutically effective amount of the pain reliever to administer to the subject to induce analgesia in the subject relative to the amount of the pain reliever to administer to the standard to induce analgesia in the standard.
Hence, the present invention extends to a method for determining a therapeutically effective amount of pain reliever to administer to a subject in order to induce analgesia in the subject relative to a therapeutically effective amount of the pain reliever to administer to a standard in order to induce analgesia in the standard wherein the method comprises the steps of removing a bodily sample comprising a first and second allele of a human mu opioid receptor gene from the subject, and determining whether the first allele comprises a human mu opioid receptor gene comprising a DNA sequence having at least one variation in SEQ ID NO:1, wherein the variation comprises:
A118G; or
C17T.
The presence of at least one variation in the human mu opioid receptor gene of the first allele from the bodily sample is expected to be indicative of the therapeutically effective amount of pain reliever to administer to the subject to induce analgesia in the subject relative to the therapeutically effective amount of pain reliever to administer to the standard to induce analgesia in the standard, wherein the standard comprises a first allele comprising a human mu opioid receptor gene comprising a DNA sequence of SEQ ID NO:1.
Moreover, the present invention further extends to a method for determining a therapeutically effective amount of pain reliever to administer to a subject in order to induce analgesia in the subject relative to a therapeutically effective amount of pain reliever to administer to a standard to induce analgesia therein, further comprising the steps of removing a bodily sample comprising a first and second allele comprising a human mu opioid receptor gene from the subject, and determining whether the second allele of the bodily sample comprises a human mu opioid receptor gene comprising a DNA sequence comprising at least one variation in SEQ ID NO:1, wherein the variation comprises:
A118G; or
C17T.
The presence of at least one variation in the human mu opioid receptor gene of the first and/or second allele of the bodily sample is expected to be indicative of the therapeutically effective amount of pain reliever to administer to the subject to induce analgesia therein relative to the amount of pain reliever to administer to a standard to induce analgesia therein, wherein the first and second alleles of the standard comprise a human mu opioid receptor gene comprising a DNA sequence of SEQ ID NO:1.
More particularly, a variation of the human mu opioid receptor gene of the first and/or second allele from the bodily sample taken from the subject, comprising a DNA sequence comprising a variation in SEQ ID NO:1, wherein the variation comprises A18G, is expected to be indicative of a decreased susceptibility to pain in the subject relative susceptibility of pain in the standard. Consequently, the subject requires a decreased therapeutically effective amount of pain reliever in order to induce analgesia therein relative to the therapeutically effective amount of pain reliever needed to induce analgesia in the standard.
In contrast, a variation of the DNA sequence of the human mu opioid receptor gene of the first and/or second allele from the bodily sample taken from the subject, comprising C17T, is expected to be indicative of an increased susceptibility to pain in the subject relative to the susceptibility to pain in the standard. Hence, the therapeutically effective amount of pain reliever to administer to the subject in order to induce analgesia therein is greater than the therapeutically effective amount of pain reliever to administer to the standard to induce analgesia therein.
In another embodiment, the present invention extends to determining a therapeutically effective amount of pain reliever to administer to a subject in order to induce analgesia in the subject, by examining a bodily sample from a subject for the presence of a variant human mu opioid receptor comprising an amino acid sequence having a variation in SEQ ID NO:2. More specifically, the present invention extends to a method for determining a therapeutically effective amount of pain reliever to administer to a subject in order to induce analgesia in the subject, relative to a therapeutically effective amount of pain reliever to administer to a standard in order to induce analgesia in the standard, comprising the steps of removing a bodily sample comprising a human mu opioid receptor from the subject, and determining whether the human mu opioid receptor present in the sample comprises an amino acid sequence having at least one variation in SEQ ID NO:2, wherein the variation comprises:
Asn40Asp or conserved variants thereof; or
Ala6Val or conserved variants thereof,
such that the presence of at least one variation is expected to be indicative of the therapeutically effective amount of pain reliever to administer to the subject to induce analgesia therein relative to the therapeutically effective amount of pain reliever to administer to induce analgesia in the standard, wherein the human mu opioid receptor of the standard comprises an amino acid sequence of SEQ ID NO:2.
In particular, the presence of a variant human mu opioid receptor comprising an amino acid sequence having a variation in SEQ ID NO:2, wherein the variation comprises Asn40Asp or conserved variants thereof in the sample from the subject, is expected to be indicative of a decreased therapeutically effective amount of pain reliever to administer to the subject to induce analgesia therein relative to the therapeutically effective amount of pain reliever to administer to the standard in order to induce analgesia therein.
In contrast, the presence of a variant human mu opioid receptor in the sample from the subject, wherein the receptor comprises an amino acid sequence have a variation in SEQ ID NO:2, wherein the variation comprises Ala6Val or conserved variants thereof, is expected to be indicative of an increased therapeutically effective amount of pain reliever to administer to the subject in order to induce analgesia therein relative to the therapeutically effective amount to administer to the standard to induce analgesia therein.
Examples of pain relievers having applications in this embodiment of the present invention include, but are not limited to, morphine, codeine, dihydromorphin, meperidine, methadone, fentanyl and its congeners, butorphenol, nalbuphine, LAAM, or propoxyphine, to name only a few.
Furthermore, the present invention extends to a method for determining a therapeutically effective amount of a therapeutic agent for treating at least one addictive disease to 15 administer to a subject suffering from at least one addictive disease, relative to a therapeutically effective amount of the therapeutic agent to administer to a standard suffering from the at least one addictive disease. As a result, the dosage of therapeutic agent administered to an addict can be xe2x80x9ctailoredxe2x80x9d to the addict""s needs based upon the addict""s genotype. An example of such a method comprises the steps of removing a bodily sample from the subject, wherein the bodily sample comprises a first and second allele of the human mu opioid receptor gene, and determining whether the first allele comprises a DNA sequence having at least one variation in SEQ ID NO:1, wherein the variation comprises:
A118G; or
C17T.
The presence of the at least one variation in the human mu opioid receptor gene of the first allele in the bodily sample from the subject is related to the therapeutically effective amount of therapeutic agent to administer to the subject to treat the subject""s at least one addictive disease, relative to the therapeutically effective amount of the therapeutic agent to administer to the standard suffering from the at least one addictive disease, wherein the first and second allele of the standard comprise a human mu opioid receptor gene comprising a DNA sequence of SEQ ID NO:1.
Furthermore, a method for determining a therapeutically effective amount of therapeutic agent to administer to a subject suffering from at least one addictive disease may further comprise an additional step of determining whether the second allele of the bodily sample taken from the subject comprises a human mu opioid receptor gene comprises a DNA sequence having at least one variation in SEQ ID NO:1, wherein the at least one variation comprises:
A118G; or
C17T.
Such a variation in the first and/or second allele of the bodily sample is expected to be indicative of the therapeutically effective amount of the therapeutic agent to administer to the subject to treat the at least one addictive disease of the subject relative to the therapeutically effective amount of the therapeutic agent to administer to the standard suffering from the at least one addictive disease.
The presence of a human mu opioid receptor gene comprising a DNA sequence having at least one variation in SEQ ID NO:1 in the first and/or second alleles of the bodily sample taken from the subject, wherein the variation comprises A118G is expected to be indicative of a decreased therapeutically effective amount of the therapeutic agent to administer to the subject to treat the at least one addictive disease of the subject, relative to the therapeutically effective amount of the therapeutic agent to administer to the standard suffering from the at least one addictive disease, wherein the two alleles of the standard comprise a human mu opioid receptor gene comprising a DNA sequence of SEQ ID NO:1.
Furthermore, the presence of a human mu opioid receptor gene comprising a DNA sequence having at least one variation of SEQ ID NO:1 in the first and/or second allele of the bodily sample taken from the subject, wherein the variation comprises C17T, is expected to be indicative of an increased therapeutically effective amount of the therapeutic agent to administer to treat the at least one addictive disease of the subject relative to the therapeutically effective amount of the therapeutic agent to administer to the standard suffering from the at least one addictive disease to treat the at least one addictive disease in the standard, wherein the alleles of the standard comprise a human mu opioid receptor gene comprising a DNA sequence of SEQ ID NO:1.
In another embodiment, the present invention extends to determining a therapeutically effective amount of a therapeutic agent for treating at least one addictive disease to administer to a subject suffering from at least one addictive disease by examining a bodily sample from a subject for the presence of a variant human mu opioid receptor comprising an amino acid sequence having at least one variation in SEQ ID NO:2. More specifically, the present invention extends to a method for determining a therapeutically effective amount of therapeutic agent for treating at least one addictive disease to administer to a subject suffering from the at least one addictive disease, relative to a therapeutically effective amount of the therapeutic agent to administer to a standard suffering from the at least one addictive disease, wherein the method comprises the steps of removing a bodily sample comprising a human mu opioid receptor from the subject, and determining whether the human mu opioid receptor present in the sample comprises an amino acid sequence having at least one variation in SEQ ID NO:2, wherein the variation comprises:
Asn40Asp or conserved variants thereof; or
Ala6Val or conserved variants thereof.
The presence of at least one variation in the human mu opioid receptor of the bodily sample is expected to be indicative of therapeutically effective amount of the therapeutic agent to administer to the subject to treat the at least one addictive disease of the subject relative to the therapeutically effective amount of the therapeutic agent to administer to the standard suffering from the at least one addictive disease, wherein the human mu opioid receptor of the standard comprises an amino acid sequence of SEQ ID NO:2.
In particular, the presence of a variant human mu opioid receptor comprising an amino acid sequence having at least one variation in SEQ ID NO:2 comprising Asn40Asp or conserved variants thereof in the bodily sample of the subject is expected to be indicative of a decreased therapeutically effective amount of the therapeutic agent to administer to the subject to treat the at least one addictive disease in the subject relative to the therapeutically effective amount of the therapeutic agent to administer to the standard to treat the at least one addictive disease therein.
Furthermore, the presence of a variant human mu opioid receptor comprising an amino acid sequence having at least one variation in SEQ ID NO:2, wherein the variation comprises Ala6Val or conserved variants thereof in the bodily sample of the subject is expected to be indicative of an increased therapeutically effective amount of the therapeutic agent to administer to the subject in order treat the at least one addictive disease in the subject relative to the therapeutically effective amount of the therapeutic agent to administer to the standard suffering from the at least one addictive disease. Examples of at least one addictive disease includes, but is not limited to opioid addiction, cocaine addiction or addiction to other psychostimulants, nicotine addiction, barbiturate or sedative hypnotic addiction, anxiolytic addiction, or alcohol addiction. Furthermore, examples of therapeutic agents having applications of the present invention include methadone, LAAM, maltrexone, or buprinorphine, to name only a few.
Furthermore, the present invention extends to a method for diagnosing a disease or disorder related to a physiological function regulated by the HPA or HPG axes of the neuroendocrine system. The HPA and HPG axes play an important role in regulation of numerous physiological activities such as reproductive and sexual function, gastrointestinal motility, immune response to an antigen, or an ability to withstand stress. Furthermore, the HPA and HPG axes exert such regulatory control via the production of endogenous opioids that interact with opioid in many locations of the body. In particular, in the HPG axis, the mu opioid receptor is centrally involved in tonic regulation of the luteinizing hormone, particularly in its pulsatile release. Furthermore, in the HPA axis, the mu opioid receptor modulates corticotropin releasing factor/hormone (CRF or CRH) in the hypothalamus which in turn modulates production of pro-opiomelanocortin (POMC) in the pituitary which is processed into several active peptides such as ACTH, which stimulates the adrenal cortex to release the stress hormone cortisol in humans, which in turn provides the stress response to environmental stimuli. Furthermore, modulated mu opioid receptor activity can lead to modulation of most cellular and humoral immunity including that mediate through T cells, B cells, cytokines, and chemokines. The pathophysiology of immune disorders may therefore be influenced by pharmacotherapies that modulate the activity of the mu opioid receptor. Moreover, gastrointestinal motility is modulated by modulation of opioid receptor treatment, and diagnosis of a disease or disorder related to gastrointestinal motility (e.g. constipation) may be facilitated by knowledge of intrinsic mu opioid receptor motility.
Applicants have discovered that the binding affinity of an opioid receptor, such as a mu opioid receptor with an endogenous opioid ligand, such as xcex2-endorphin, is expected to modulate such physiological activities. Hence, the binding affinity of variant mu opioid receptors explained above, for endogenous opioid ligands such as xcex2-endorphin, is expected to modulate those physiological activities regulated by the HPA and HPG axes relative to those physiological activities in a standard having mu opioid receptors produced from the predominant or xe2x80x9cmost commonxe2x80x9d allele of the mu opioid receptor gene comprising a DNA sequence of SEQ ID NO:1. As the result, the present invention extends to a method of diagnosing a disease or disorder related to a physiological function regulated by the HPA or HPG axes. Examples of physiological functions regulated by the HPA and the HPG include, but are not limited to sexual or reproductive functions, gastrointestinal motility, immune response, or ability to withstand stress. Such a method comprises the steps of removing a bodily sample comprising a first and second allele of a human mu opioid receptor gene from the subject, and determining whether the first allele comprises a human mu opioid receptor gene comprising a DNA sequence having at least one variation in SEQ ID NO:1, wherein the variation comprises:
A118G; or
C17T.
The presence of at least one variation in the human mu opioid receptor gene of the first allele is expected to be indicative of a disorder related to a physiological function regulated by the HPA or GPA, such as sexual or reproductive functions, gastrointestinal motility, immune response, and the ability to withstand stress, wherein the first allele of the standard comprises a human mu opioid receptor gene comprising a DNA sequence of SEQ ID NO:1.
Moreover, a method for diagnosing a disease or disorder related to a physiological function regulated by the HPA or GPA, as described above may further comprise the step of determining whether the second allele of the bodily sample comprises a human mu opioid receptor gene comprising a DNA sequence having at least one variation in SEQ ID NO:1, wherein the variation comprises:
A118G; or
C17T.
The presence of the at least one variation in the human mu opioid receptor gene of the second allele of the bodily sample from the subject may be expected to be indicative of a disease or disorder related to sexual and reproductive functions, gastrointestinal motility, immune response, or the ability of the subject to withstand stress.
At least one variation in the human mu opioid receptor gene of the first and/or second allele of the bodily sample taken from the subject, wherein the variation comprises A118G, is expected to be indicative of decreased HPA and HPG activity, resulting in increased sexual or reproductive functions, increased gastrointestinal motility, increased immune response, or increased ability to withstand stress relative to the levels of such function observed in a standard.
Furthermore, the presence of at least one variation comprising C17T in the human mu opioid receptor gene of the first and/or second allele of the bodily sample from the subject is expected to be indicative increased HPA or HPG activity, resulting in decreased sexual or reproductive function, decreased gastrointestinal motility, decreased immune response, or decreased ability to withstand stress relative to the levels of such function observed in a standard.
In another embodiment, the present invention extends to a method for diagnosing a disease or disorder related to a physiological function regulated by the HPA or GPA by examining a bodily sample taken from the subject for the presence of a variant human mu opioid receptor Such a method comprises the steps of removing a bodily sample comprising a human mu opioid receptor from the subject, and determining whether the human mu opioid receptor present in the sample is a variant human mu opioid receptor of the invention, i.e., comprises an amino acid sequence having at least one variation in SEQ ID NO:2, wherein the variation comprises:
Asn40Asp or conserved variants thereof; or
Ala6Val or conserved variants thereof,
such that the presence of at least one variation is expected to be indicative of a disease or disorder related to a physiological activity regulated by the HPA or HPG axes, such as sexual function or development, gastric motility, immune response, or the ability of the subject to withstand stress, relative to regulation of such activities in a standard comprising a human mu opioid receptor having an amino acid sequence of SEQ ID NO:2.
In particular, the presence of a variant human mu opioid receptor comprising an amino acid sequence having at least one variation in SEQ ID NO:2 wherein the variation comprises Asn40Asp or conserved variants thereof, is expected to be indicative decreased HPA and HPG activity, resulting in increased sexual or reproductive functions, increased gastrointestinal motility, increased immune response, or increased ability to withstand stress relative to the levels of such function observed in a standard having a mu opioid receptor comprising an amino acid sequence of SEQ ID NO:2.
Furthermore, the presence of a variant human mu opioid receptor comprising an amino acid sequence having a variation in SEQ ID NO:2, wherein the variation comprises Ala6Val or conserved variants thereof, in a bodily sample taken from a subject is expected to be indicative of increased activity of the HPA and HPG axes, resulting in decreased sexual or reproductive functions, decreased gastrointestinal motility, decreased immune response, or decreased ability to withstand stress relative to the levels of such function observed in a standard having a mu opioid receptor comprising an amino acid sequence of SEQ ID NO:2. Examples of specific diseases or disorders related to regulation of physiological functions by the HPA or HPG axes include infertility, constipation, diarrhea, decreased immune response to antigens relative to a standard, or decreased of ability to withstand stress relative to a standard.
Once a disease or disorder related to a physiological function regulated by the HPA or HPG axes has been diagnosed, it is possible for attending medical professionals treating the subject to select and administer an appropriate therapeutic agent and a therapeutically effective amount of the agent to administer to the subject to treat such a disease or disorder. Consequently, the present invention extends to a method for determining an appropriate therapeutic agent to administer to a subject suffering from a disease or disorder related to a physiological function regulated by the HPA or HPG axes, comprising removing a bodily sample from the subject, and determining the presence of at least one variant allele of a mu opioid receptor gene in the bodily sample, wherein the variant allele comprises a human mu opioid receptor gene comprising a DNA sequence having at least one variation in SEQ ID NO:1, wherein the variation comprises:
A118G; or
C17T.
The present invention further extends to a method for selecting an appropriate therapeutic agent to administer to a subject suffering from a disease or disorder related to a physiological function regulated by the HPA or HPG axes as set forth above, further comprising determining whether the bodily sample comprises a second variant allele of the mu opioid receptor gene comprising a DNA sequence having a variation in SEQ ID NO:1, wherein the variation comprises:
A118G; or
C17T.
In particular, should either or both alleles of the mu opioid receptor gene of the bodily sample comprise a DNA sequence having a variation in SEQ ID NO:1, wherein the variation comprises A118G, the mu opioid receptors of the subject are expected to have increased function relative to mu opioid receptors of a standard produced from expression of the predominant or xe2x80x9cmost commonxe2x80x9d mu opioid receptor allele comprising a DNA sequence of SEQ ID NO:1. This increased function is expected to result in decreased function of the HPA and HPG axes. Hence, an appropriate therapeutic agent for treating a disease or disorder related to decreased activity of the HPA or HPG axes, such as diarrhea can be selected.
In contrast, a human mu opioid receptor produced from expression of a variant allele of a mu opioid receptor gene comprising a variation in SEQ ID NO:1, wherein the variation comprises C17T is expected to have decreased activity relative to a mu opioid receptor produced from expression of the predominant or xe2x80x9cmost commonxe2x80x9d allele of the human mu opioid receptor gene comprising a DNA sequence of SEQ ID NO:1. This decreased activity is expected to result in higher activity of the HPA and HPG axes. As a result, a medical professional attending the subject is able to select an appropriate therapeutic agent for treating a disease or disorder related to sexual and reproductive functions, such as infertility, gastrointestinal motility, such as constipation or diarrhea, decreased immune response towards antigens relative to immune response in a standard, or decreased ability to withstand stress relative to ability to withstand stress in a standard.
The present invention further extends to commercial test kits suitable for use by a medical professional to determine whether either or both alleles of a bodily sample taken from a subject comprise a DNA sequence having at least one variation in SEQ ID NO:1, wherein the variation comprises:
A118G; or
C17T.
Commercial test kits of the present invention have applications in determining susceptibility of pain in the subject relative to a standard. Such kits can also be used to determine a subject""s increased or decreased susceptibility to at least one addictive disease relative to susceptibility to at least one addictive disease in a standard. Also a therapeutically effective amount of pain reliever to administer to the subject in order to induce analgesia in the subject relative to a therapeutically effective amount of pain reliever to administer to a standard to induce analgesia in the standard can be determined. Moreover, a test kit of the present invention has applications in determining a therapeutically effective amount of therapeutic agent for treating at least one addictive disease to administer to a subject suffering from the at least one addictive disease, relative to a therapeutically effective amount of therapeutic agent to administer to a standard suffering from at least one addictive disease. Furthermore, test kits of the invention have applications in diagnosing a disease or disorder related to a physiological condition regulated by the HPA or HPG axes of the neuroendocrine system, and in selecting an appropriate therapeutic agent for treating such a disease or disorder, along with a therapeutically effective amount of agent to administer to the subject. A standard as used herein comprises two alleles of a human mu opioid receptor gene comprising a DNA sequence of SEQ ID NO:1.
Furthermore, a commercial test kit of the present invention can also be used to determine the presence of an isolated variant allele of a human mu opioid receptor gene of the present invention in a bodily sample removed from a subject, which can serve as a genetic marker. As explained above, the predominant or xe2x80x9cmost commonxe2x80x9d allele of a human mu opioid receptor gene found in the population comprises a DNA sequence of SEQ ID NO:1. Hence a variant allele comprising a DNA sequence having a variation in SEQ ID NO:1, wherein the variation comprises:
G24A;
G779A; or
G942A,
or combinations thereof, can be detected in the bodily sample with a commercial kit of the invention.
Other variant alleles of the human mu opioid receptor gene of the present invention can be detected with a commercial test kit of the present invention. For example, an isolated variant allele of a human mu opioid receptor gene detectable with a commercial kit of the present invention, comprises a DNA sequence having at least two variations in SEQ ID NO:1, wherein the variations comprise:
A118G;
C17T;
G24A;
G779A; or
G942A.
Accordingly, a commercial test kit may be prepared for determining the presence of at least one variation in a human mu opioid receptor gene of either or both alleles in a bodily sample taken from a subject, wherein the commercial test kit comprises:
a) PCR oligonucleotide primers suitable for detection of an allele comprising a human mu opioid receptor gene having a DNA sequence with a variation in SEQ ID NO:1;
b) other reagents; and
c) directions for use of the kit.
The present invention further extends to commercial test kits capable of detecting a variant human mu opioid receptor in a bodily sample taken from a subject. Examples of variant human mu opioid receptors that can be detected with a kit of the present invention comprise:
a variant human mu opioid receptor comprising an amino acid sequence having a variation in SEQ ID NO:2, wherein the variation comprises Arg260His or conserved variants thereof; or
a variant human mu opioid receptor comprising an amino acid sequence having at least two variations in SEQ ID NO:2, wherein the variations comprise:
Asn40Asp or conserved variants thereof,
Ala6Val or conserved variants thereof; or
Arg260His or conserved variants thereof.
Moreover, a commercial test kit of the present invention can be used to determine: susceptibility to pain in the subject relative to susceptibility to pain in a standard; a therapeutically effective amount of pain reliever to administer to a subject to induce analgesia in the subject relative to a therapeutically effective amount of pain reliever to administer to a standard to induce analgesia in the standard; a therapeutically effective amount of therapeutic agent for treating at least one addictive disease to administer to a subject suffering from at least one addictive disease, relative to a therapeutically effective amount of therapeutic agent to administer to a standard suffering from the at least one addictive disease; diagnosing a disease or disorder related to a physiological condition regulated by the HPA or HPG axes of the neuroendocrine system, or selecting an appropriate therapeutic agent for treating such a disease or disorder, along with a therapeutically effective amount of such agent to administer to the subject. Accordingly, the present invention extends to a commercial test kit having applications set forth above, comprising a predetermined amount of at least one detectably labeled immunochemically reactive component having affinity for a variant human mu opioid receptor;
(b) other reagents; and
(c) directions for use of the kit.
In a further variation, the test kit may be prepared and used for the purposes stated above, which operates according to a predetermined protocol (e.g. xe2x80x9ccompetitive,xe2x80x9d xe2x80x9csandwich,xe2x80x9d xe2x80x9cdouble antibody,xe2x80x9d etc.), and comprises:
(a) a labeled component which has been obtained by coupling the human mu opioid receptor of a bodily sample to a detectable label;
(b) one or more additional immunochemical reagents of which at least one reagent is a ligand or an immobilized ligand, which ligand comprises:
(i) a ligand capable of binding with the labeled component (a);
(ii) a ligand capable of binding with a binding partner of the labeled component (a);
(iii) a ligand capable of binding with at least one of the component(s) to be determined; or
(iv) a ligand capable of binding with at least one of the binding partners of at least one of the component(s) to be determined; or
(c) directions for the performance of a protocol for the detection and/or determination of one or more components of an immunochemical reaction between the human mu opioid receptor gene of the present invention and a specific binding partner thereto.
Accordingly, it is an object of the present invention to provide heretofore unknown variations the DNA sequence of the human mu opioid receptor gene wherein the variations can be used to map the locus of the human mu opioid receptor gene.
It is yet another object of the present invention to use heretofore unknown polymorphisms of an allele of the human mu opioid receptor gene as markers for any kind of disorder related to the human mu opioid receptor, such as an addictive disease, pain, or markers for genes.
It is another object of the present invention to provide nucleotides, optionally detectably labeled, hybridizable under standard hybridization conditions to variant alleles of the human mu opioid receptor gene disclosed herein, as well as polypeptides produced from the expression of the variant alleles and nucleotides hybridizable thereto under standard hybridization conditions.
It is yet another object of the present invention to provide antibodies, optionally detectably labeled, having immunogens comprising polypeptides produced from the expression of variant alleles of human mu opioid receptor gene, or expression of isolated nucleic acid molecules hybridizable under standard hybridization conditions to variant alleles disclosed herein.
It is another object of the present invention to gain insight into a subject""s susceptibility to pain. This insight can be used to determine a therapeutically effective dose of pain reliever to administer to the subject to induce analgesia therein relative to the therapeutically effective amount of pain reliever administered to a standard to induce analgesia therein, wherein the standard comprises two alleles of the human mu opioid receptor gene comprising a DNA sequence of SEQ ID NO:1, or a variant human mu opioid receptor comprising an amino acid sequence of SEQ ID NO:2.
Such information can be used to tailor a regimen for treating a subject suffering from at least one addictive disease, relative to the therapeutically effective amount of therapeutic agent administered to a standard suffering from at least one addictive disease.
It is yet another object of the present invention to provide commercial test kits for attending medical professionals to determine the presence of variant alleles of a human mu opioid receptor gene in a bodily sample taken from a subject. The results of such testing can then be used to determine the subject""s susceptibility to pain, susceptibility to at least one addictive disease, determining a therapeutically effective amount of pain reliever to administer to the subject in order to induce analgesia, or determining a therapeutically effective amount of therapeutic agent for treating at least one addictive disease to administer to the subject.
It is an object of the present invention to determine the activity of a mu opioid receptor in a subject, and use such information to diagnose a disease or disorder related to sexual or reproductive function, gastrointestinal motility, immune response, or ability to withstand stress, wherein variant alleles of the mu opioid receptor gene when expressed produce variant mu opioid receptors having activity different from a mu opioid receptor produced from the predominant or xe2x80x9cmost commonxe2x80x9d allele of the mu opioid receptor comprising a DNA sequence of SEQ ID NO:1.
It is another object of the present invention to employ Applicants"" discovery of a correlation between the activity of a mu opioid and its impact the neuroendocrine system, and particularly on levels of hormones within the body. As a result, the level of activity of the mu opioid receptor effects sexual or reproductive function, gastrointestinal motility, immune response, or ability to withstand stress. Such information can further be used select appropriate therapeutic agents to treat diseases such as infertility, constipation, or diarrhea. Further, such information can be used to select appropriate therapeutic agents to increase immune response against an antigen such as a bacterium, a virus or a tumor cell in the subject, and to treat psychiatric diseases or disorders such as obsessive compulsive disorder, schizophrenia, or depression.
It is yet another object of the present invention to provide commercial detecting variant alleles of the human mu opioid receptor gene or the presence of a variant human mu opioid receptor in a bodily sample taken from a subject. The results of such tests can then be used to gain incite into a subject""s ability to withstand pain, susceptibility to addiction, to diagnose a disease or disorder related to a physiological function regulated by the HPA or HPG axes such as sexual and reproductive functions, gastrointestinal motility, immune response, and the ability of the subject to withstand stress.
These and other aspects of the present invention will be better appreciated by reference to the following drawings and Detailed Description.